Rationale: Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking.Objective: To test the hypotheses that OxLDL triggers reverse translocation of mitochondrial arginase 2 (Arg2) to cytosol and Arg2 activation, and that this process is dependent on mitochondrial processing peptidase, lectin-like OxLDL receptor-1 receptor, and rho kinase. Methods and Results: Conclusions

The ability to understand and regulate human vasculature development and differentiation has the potential to benefit patients suffering from a variety of ailments, including cardiovascular disease, peripheral vascular disease, ischemia, and burn wounds. Current clinical treatments for vascular-related diseases commonly use the grafting from patients of autologous vessels, which are limited and often damaged due to disease. Considerable progress is being made through a tissue engineering strategy in the vascular field. Tissue engineering takes a multidisciplinary approach seeking to repair, improve, or replace biological tissue function in a controlled and predictable manner. To address the clinical need to perfuse and repair damaged, ischemic tissue, one approach of vascular engineering aims to understand and promote the growth and differentiation of vascular networks. Vascular tissue engineered constructs enable the close study of vascular network assembly and vessel interactions with the surrounding microenvironment. Scaffold platforms provide a method to control network development through the biophysical regulation of different scaffold properties, such as composition, mechanics, dimensionality, and so forth. Following a short description of vascular physiology and blood vessel biomechanics, the key principles in vascular tissue engineering are discussed. This review focuses on various biodegradable scaffold platforms and demonstrates how they are being used to regulate, promote, and understand angiogenesis and vascular network formation.

A significant challenge in oncology is the need to develop in vitro models that accurately mimic the complex microenvironment within and around normal and diseased tissues. Here, we describe a self-folding approach to create curved hydrogel microstructures that more accurately mimic the geometry of ducts and acini within the mammary glands, as compared to existing three-dimensional block-like models or flat dishes. The microstructures are composed of photopatterned bilayers of poly (ethylene glycol) diacrylate (PEGDA), a hydrogel widely used in tissue engineering. The PEGDA bilayers of dissimilar molecular weights spontaneously curve when released from the underlying substrate due to differential swelling ratios. The photopatterns can be altered via AutoCAD-designed photomasks so that a variety of ductal and acinar mimetic structures can be mass-produced. In addition, by co-polymerizing methacrylated gelatin (methagel) with PEGDA, microstructures with increased cell adherence are synthesized. Biocompatibility and versatility of our approach is highlighted by culturing either SUM159 cells, which were seeded postfabrication, or MDA-MB-231 cells, which were encapsulated in hydrogels; cell viability is verified over 9 and 15 days, respectively. We believe that self-folding processes and associated tubular, curved, and folded constructs like the ones demonstrated here can facilitate the design of more accurate in vitro models for investigating ductal carcinoma.

Effects of 3D confinement on cellular growth and matrix assembly are important in tissue engineering, developmental biology, and regenerative medicine. Polydimethylsiloxane wells with varying anisotropy are microfabicated using soft‐lithography. Microcontact printing of bovine serum albumin is used to block cell adhesion to surfaces between wells. The orientations of fibroblast stress fibers, microtubules, and fibronectin fibrils are examined 1 day after cell seeding using laser scanning confocal microscopy, and anisotropy is quantified using a custom autocorrelation analysis. Actin, microtubules, and fibronectin exhibit higher anisotropy coefficients for cells grown in rectangular wells with aspect ratios of 1:4 and 1:8, as compared to those in wells with lower aspect ratios or in square wells. The effects of disabling individual cytoskeletal components on fibroblast responses to anisotropy are then tested by applying actin or microtubule polymerization inhibitors, Rho kinase inhibitor, or by siRNA‐mediated knockdown of AXL or cofilin‐1. Latrunculin A decreases cytoskeletal and matrix anisotropy, nocodazole ablates both, and Y27632 mutes cellular polarity while decreasing matrix anisotropy. AXL siRNA knockdown has little effect, as does siRNA knockdown of cofilin‐1. These data identify several specific cytoskeletal strategies as targets for the manipulation of anisotropy in 3D tissue constructs.

Oxidized Low density lipoprotein (OxLDL) acts via its receptor LOX‐1 and elicits endothelial dysfunction that is considered a hallmark for initiation and progression of atherosclerosis. However, the precise mechanism by which this occurs is still unclear and is the main focus of current study. Our time course, subcellular fractionation, and immunofluorescence data support our hypothesis that Arg2 is a dual‐targeted single gene and single mRNA product found in both mitochondrial and cytosolic fractions of HAEC ‐ a novel pattern of effector system activation in mammalian vascular injury. These data indicate that OxLDL‐mediated activation of Arg2 in HAEC and in mouse aortic intima occurs via post‐translational modification and subcellular translocation of pre‐existing pools of Arg2 from mitochondria to the cytosol. Accumulated Arginase 2 in cytosol diminishes the concentration of cytosolic L‐arginine and leads to eNOS uncoupling. OxLDL‐evoked changes in cytosolic Arg2 activity are muted by Rho‐kinase inhibition, and do not occur at all in LOX‐1 null endothelial cells. In addition, inhibition or siRNA knockdown of the mitochondrial processing peptidase (MPPα) reduces the cytosolic abundance and activity of Arg2 following OxLDL stimulation. Blocking MPP with the biochemical inhibitor O‐phenanthroline also attenuates OxLDL‐mediated changes in HAEC production of ROS and NO. Finally, knockdown of MPPα by adenovirally delivered MPP shRNA interference in isolated mice aortas was associated with improved endothelium‐dependent vascular relaxation as assessed by wire myography following OxLDL exposure, when compared with aortas treated with scrambled shRNA. Hence, inhibition of Arg2 translocation to cytosol by targeting MPP could provide novel therapeutic avenues for the treatment of atherosclerosis and other cardiovascular diseases. Grant Funding Source: NIH, AHA

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